The dopamine transporter (DAT) is the primary mechanism by which extracellular dopamine (DA) is cleared from the synaptic space. As such it performs a key role in terminating dopaminergic neurotransmission and in regulating the concentration of DA available for binding to pre- and post-synaptic receptors. DATs are targets for numerous psychoactive drugs including abused compounds such as cocaine, and amphetamine (AMPH), and therapeutic agents such as Ritalin and Wellbutrin. Dysrgulation of DAT, which would lead to abnormal levels of DA, is speculated to occur in dopaminergic diseases such as Parkinson's disease and schizophrenia, although mechanisms are not known. DATs are highly regulated proteins, with current evidence implicating the involvement of numerous protein kinases including Protein Kinase C and Calcium- Calmodulin Dependent Protein Kinase. While we know that DATs are metabolically phosphorylated, we do not know the precise sites on the protein that are modified, the kinases or phosphatases that catalyze phosphate addition or removal, or the molecular basis by which phosphorylation effects functional regulation. There is also only marginal understanding of the relationship of this process to abused or therapeutic drugs. The long term goal of this project is to clarify the precise mechanisms by which DAT is regulated by phosphorylation. The specific aims are to elucidate the kinases and phosphatases involved, the amino acids modified, and the subcellular regions where these processes occur;determine how phosphorylation at specific sites impacts AMPH-stimulated efflux;and to determine how phosphorylation is impacted by DAT blockers. The completion of these studies will provide a much more precise understanding of DAT phosphorylation and how this process could be related to dysregulation of DAT in dopaminergic diseases and drug abuse. PUBLIC HELATH RELEVANCE: The completion of these studies will provide a much more precise understanding of DAT phosphorylation and its role in processes induced by amphetamine and dopamine transport blockers. This information may thus be useful in elucidating novel therapeutic targets for treating drug addiction, and help to clarify potential links to DAT dysregulation in dopaminergic disorders such as Parkinson's disease and depression.